The present invention relates to a method for detecting a double-stranded DNA having a defined nucleotide sequence by using a single-stranded probe.
Screening a DNA having a defined sequence is a technique for use in molecular biology and in the clinical test field. In particular, it is a basic technique for use in gene cloning and detecting genetic disorders. As a conventional DNA detection method, a Southern blot technique is widely used which detects a single-stranded DNA (hereinafter ssDNA) having a defined nucleotide sequence by binding a labeled probe to the ssDNA sample which has been prepared by denaturing treatment. On the other hand, as a method enabling direct detection of the double-stranded DNA (hereinafter dsDNA) having a defined sequence in a dsDNA sample without the denaturing treatment, the following techniques are known in the prior art.
A first method is characterized in that an oligonucleotide probe is allowed to bind to a target DNA having a defined nucleotide sequence via recA protein to form a complex consisting of these three substances (hereinafter, referred to as "recA recombinant intermediate")(Cheng et al., J. Biol. Chem, 263, 15110-15117, 1988). In this method, after a recA protein digestion reaction, which is required for subjecting the dsDNA sample to a separation gel, a photocrosslinking reaction is performed to prevent the binding of the oligonucleotide probe to the target dsDNA from being unstable. In this manner, stable binding between the target dsDNA and the probe is attained. The ultraviolet irradiation during the photocrosslinking reaction has drawbacks not only in producing a non-specific binding of the probe to the target DNA but also in damaging the target DNA itself.
To overcome the problems in the prior art, the present inventors have disclosed a method for detecting a specific nucleotide sequence in the double-stranded nucleic acid without causing damage to nucleic acids, in Japanese Patent Publication No. 8-54278. In the second method mentioned above, instead of binding the probe to the target dsDNA by the photocrosslinking reaction, a probe is fixed onto the target DNA in the form of loop under mild conditions using enzyme reactions. Thus, this method is free from the drawback of damaging the target dsDNA. However, in this method, a probe-bound DNA finally produced does not fully represent the size and shape of the target dsDNA molecule correctly in the detection step with the separation gel.